Preparing frozen stock of competent DH5 cells.
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Streak cells from the frozen stock on LB plate without antibiotic. Incubate
overnight at 37C.
Transfer 4 or 5 well-isolated colonies into 1ml LB containing 20mM MgSO4,
vortex and dilute in 30-100ml of LB with 20mM MgSO4 in a 1-liter flask.
Grow cells for 2.5-3 hrs at 37C to the OD600 ~0.4-0.5.
Transfer the cells to sterile, ice-cold centrifuge tube (for SS-34 Sorvall rotor), cool
for 10min in ice-water bath. Spin the cells for 5min at 2000g.
Decant the media from the cell pellets. Stand the tubes in inverted position to
allow last traces of media to drain away.
Resuspend the cells by swirling and gentle vortexing in 20ml ice-cold FSB
buffer*.
Spin the cells for 5min at 2000g. Decant the media from the cell pellets. Stand the
tubes in inverted position to allow last traces of buffer to drain away.
Resuspend the cells by swirling and gentle vortexing in 4ml ice-cold FSB buffer.
Add 140l of DMSO per 4ml of resuspended cells. Mix gently by swirling and
store the suspension on ice for 15min.
Add an additional140l of DMSO to each suspension. Mix gently by swirling and
return to ice bath.
Working quickly, dispense 25l aliquots of the suspensions into chilled, sterile
eppendorf tubes. Immediately snap-freeze the cells in liquid nitrogen. Store the
cells in -70C.
When needed, remove the cells from the freezer, thaw by holding the tube in the
palm of your hand. Just as cells thaw, transfer the tube on ice and let stand for
10min. Use 0.5ng of supercoiled DNA to transform 25l of cells.
Preparation of 1L of FSB.
Reagent
1M Potassium acetate pH7.5
MnCl2 x 4H2O
CaCl2 x 2 H2O
KCl
Hexaamminecobalt chloride
Glycerol
Amount required/liter
10ml
8.91g
1.47g
7.46g
0.80g
100ml
Final concentration
10mM
45mM
10mM
100mM
3mM
10%
1M Potassium acetate is prepared by dissolving 9.82g of potassium acetate in 90ml of
Milli-Q water and adjusting pH to7.5 with 2M acetic acid and then bringing the volume
to 100ml. 10ml aliquots are stored at -20C.
Prepare the FSB by mixing all the components with 800ml of Milli-Q water and adjusting pH to
6.4 with 0.1N HCl. Do not add base if overshoot – discard and start over. Bring the volume to 1
liter, filter the FSB through 0.45m filter to sterilize. 40ml aliquots should be stored at 4C.
During the storage, the pH will drift to 6.1-6.2 and then will stabilize